A. Field of the Invention
This invention relates to the production of stably-transformed insect cell lines capable of continuous expression of a selected foreign gene product.
B. Description of the Related Art
Baculovirus expression vectors (BEVs) have become extremely important tools for the expression of foreign genes, both for basic research and for the production of proteins with direct clinical applications in human and veterinary medicine (W. Doerfler, Curr. Too. Microbiol. Immunol., 131:51-68 (1968); V. A. Luckow and M. D. Summers, Bio/Technology. 6:47-55 (l988a); L. K. Miller, Annual Review of Microbiol., 42:177-199 (1988); M. D. Summers, Curr. Communications in Molecular Biology. Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1988)). BEVs are recombinant insect viruses in which the coding sequence for a chosen foreign gene has been inserted behind the promoter in place of the nonessential viral gene, polyhedrin (Smith and Summers, U.S. Pat. No. 4,745,051).
Baculovirus genes are expressed in a sequential, temporally-regulated fashion during one or more of four different phases of the viral replication cycle (P. D. Friesen and L. K. Miller, Curr. Too. Microbiol. Immunol., 131:31-49 (1986); L. A. Guarino, CRC Press, (1989) [in press]). Therefore, different baculovirus genes may be classified as immediate-early (.alpha.), delayed-early (.beta.), late (.gamma.), or very late (.delta.), according to the phase of the viral infection during which they are expressed. The expression of these genes occurs sequentially, probably as the result of a "cascade" mechanism of transcriptional regulation. Thus, the immediate-early genes are expressed immediately after infection, in the absence of other viral functions, and one or more of the resulting gene products induces transcription of the delayed-early genes. Some delayed-early gene products, in turn, induce transcription of late genes, and finally, the very late genes are expressed under the control of previously expressed gene products from one or more of the earlier classes. One relatively well-defined component of this regulatory cascade is IEl, an immediate-early gene of Autographa californica nuclear polyhedrosis virus (AcMNPV). IEl is expressed in the absence of other viral functions and encodes a product that stimulates the transcription of several genes of the delayed-early class, including the 39K gene (L. A. Guarino and M. D. Summers, J. Virol., 57:563-571 (1986a); J. Virol., 61:2091-2099 (1987)), as well as late genes (L. A. Guarino and M. D. Summers, Virol., 162:444-451 (1988)). However it is believed that the immediate-early genes are not dependent upon other viral gene for expression.
The polyhedrin gene is classified as a very late gene. Therefore, transcription from the polyhedrin promoter requires the previous expression of an unknown, but probably large number of other viral and cellular gene products. Because of this, state-of-the-art BEVs, such as the exemplary BEV system described by Smith and Summers (U.S. Pat. No., 4,745,051) will express foreign genes only as a result of gene expression from the rest of the viral genome, and only after the viral infection is well underway. This represents a clear limitation to the use of existing BEVs for at least two reasons. First, infection with the essentially intact recombinant virus ultimately kills the host cell, thereby terminating its role as a "factory" for foreign protein production. Thus, prior art BEV systems are limited to gene expression in transient cell lines. Second, the ability of the host cell to process newly synthesized proteins decreases as the baculovirus infection progresses (D. L. Jarvis and M. D. Summers, Mol. Cell. Biol., 9:214-223 (1989)). Thus, gene expression from the polyhedrin promoter occurs at a time when the host cell's ability to process newly synthesized proteins is significantly diminished. As a consequence, the expression of secretory glycoproteins in BEV systems is complicated due to incomplete secretion of the cloned gene product, thereby trapping the cloned gene product within the cell in an incompletely processed form.